Heme from denatured proteins exacerbates oxidative injury to endothelial cells (EC) because of its reactive iron. As a protective response, heme is degraded by heme oxygenase (HO) to release iron which is bound by ferritin in a catalytically inactive form. Nitric oxide has been shown to induce HO and repress ferritin synthesis which could result in accumulation of catalytically active iron if heme degradation was unimpeded. We wondered how nitric oxide (NO) might affect the degradation of heme and release of catalytically active iron. Using electron spin resonance (ESR) spectroscopy, we found that nitrosylation of free heme readily occurs aerobically in endothelial microsomes but only little is formed to cytosol and no adducts formed in media. We used ESR to investigate the influence of HO on heme degradation during NO exposure. These experiments showed that nitrosylation of heme readily occurs in cells, and accumulation of non-heme iron is prevented in the presence of NO. Nitrosylation of heme was protective in cytotoxicity assays of EC exposed to hydrogen peroxide, suggesting that NO alters the interaction between intracellular heme and peroxides.